The broad, long-term objective of this study is to identify molecular targets regulating replacement tooth formation (RTF) in zebrafish that can be manipulated to treat tooth agenesis in humans. Zebrafish are the only currently available, tractable developmental model for RTF, and therefore provide a unique opportunity to identify and study signaling pathways regulating this process. Specifically, we will test the hypothesis, supported by much data from this laboratory, that alk8 signaling pathways mediate RTF. The alk8 gene, which encodes a novel type I TGF-beta receptor family member first identified in this laboratory, is expressed during, and is required for, zebrafish primary and RTF development. In the proposed studies, we will identify and perform functional characterization of Alk8 specific RT signaling partners, using previously identified laf/alk8 mutants, which display a tooth agenesis phenotype. RT rescue in laf/alk8 mutants will be monitored and quantified using highly sensitive in vivo assay for mineralized tooth formation in living zebrafish, and by in vitro analyses of fixed specimens. First, we will perform detailed molecular/genetic analyses of laf/alk8 mutants to define the temporal and tissue-specific, molecular and cellular defects leading to the tooth agenesis phenotype observed in these mutants, including microarray analysis of mutant and wt pharyngeal tooth tissues. Next, we will generate Gateway transgenic, heat-shock inducible dominant negative and constitutively active Alk8 transgenic laf/alk8 mutant lines to manipulate - exacerbate and rescue - tooth agenesis in laf/alk8 and wt siblings, establishing this approach as an in vivo gene delivery therapy for rescue of tooth agenesis. Finally, we will define the molecular interactions of Runx2, retinoblastoma (Rb), and Alk8 signaling in RTF, using both zebrafish and transgenic mouse models, based on published interactions of Rb and Runx2 in osteoblast differentiation, and on our preliminary data demonstrating supernumerary tooth formation in Rb null mice. The significance of the proposed studies includes the ability to: 1) define RTF signaling pathways using the only currently available, tractable, developmental model, the zebrafish;2) establish an in vivo gene delivery model for rescue of tooth agenesis;and 3) define, for the first time, the interactions of Rb, Runx2 and Alk8 in RTF. The successful completion of the proposed studies will provide an important entry for therapeutic treatment of tooth loss, by significantly expanding our current knowledge of molecular signals regulating RTF, providing the means to eventually establish clinically relevant therapies to rescue tooth agenesis in humans. PUBLIC HEALTH RELEVANCE: The relevance of the proposed studies is the potential to develop molecular based, gene delivery approaches for biological replacement tooth (RT) therapies in humans. The successful completion of the proposed studies will provide the foundation for therapeutic treatment of tooth loss in humans, by significantly expanding our current knowledge of molecular signals regulating RTF, providing the means to eventually establish clinically relevant therapies to rescue tooth agenesis in humans.